Construction Of Lactobacillus Casei Recombinant Strain Targeted By BVDV E2

Bovine viral diarrhea(BVD),also known as mucosal disease,is a widespread infectious disease of cattle in the world,which has caused huge assets to the livestock industry and dairy industry in many countries around the world.Bovine viral diarrhea is caused by Bovine viral diarrhea virus(BVDV).BVDV can cause a variety of clinical symptoms in cattle,including respiratory diseases,diarrhea,high fever,immune system diseases,,Reproductive disorders,persistent infections and mucosal diseases.BVDV is a single-stranded positive-strand RNA virus,which is enveloped by the capsule itself.The mature BVDV virus is spherical in shape,with a diameter of40--60 nm,a ring-shaped subunit on the surface of the capsule,and a size of about10-12 nm.The nucleocapsid is a 20-sided symmetrical capsid with a molecular weight of 14 Ku Protein composition.It belongs to the Pestivirus genus in the Flaviviridae family and is a representative of the genus Pestivirus of the Flaviviridae.The BVDV genome is about 12.5 Kb in total length.It contains a large open reading frame and can be translated into multiple proteins.It is divided into 4 structural proteins including viruses and 8 non-structural proteins.Four structural proteins are derived from the N-terminus of polyproteins: including nucleocapsid protein C and surface glycoproteins Erna,E1 and E2.Eight non-structural proteins include: Npro,p7,NS2,NS3,NS4 A,NS4B,NS5 A and NS5 B.BVDV E2 is the main determinant of the immunogenicity of BVDV virions,and itself has become an important potential target for the development of new BVDV vaccines.Monoclonal antibodies specific for BVDV E2 have clinically demonstratedsignificant virus neutralizing capacity for both types of BVDV-1 and BVDV-2.These conclusions provide strong support for the development of a new vaccine against BVDV E2.Therefore,BVDV E2 protein is the main research object of new vaccines.As a probiotic,Lactobacillus casei can be colonized in the bovine intestines.It plays a dual role as an adjuvant and carrier itself,it can significantly enhance the ability of cells and systemic immune system to resist foreign antigens,so they are more attractive as vaccine carriers.Currently,Lactobacillus casei is considered to be a more effective live vaccine carrier than Salmonella and Listeria.Due to the ability to enhance the host's immune response,through recombination,the internal adjuvants and cytokines of Lactobacillus casei can induce the production of specific expression of reactive antigens.Therefore,Lactobacillus casei has received more attention and application as a heterologous expression molecule.Lactobacillus casei as a carrier has been shown to significantly improve the survival rate of animals,control and reduce animal weight loss,reduce liver and large intestine damage,and has better protective efficacy than traditional inactivated vaccines.In this study,we first constructed the L.casei UPP knockout plasmid containing UPP upstream homology arm(UPP-u)-slpA-BVDV E2-GFP-pgsA-rrnBT1T2-UPP downstream homology arm(UPP-d)fusion protein expression cassette.After the construction was completed,the genotype identification,the sequence consistency with the genes in the NCBI library reached 99%.Secondly,the UPP knockout plasmid is introduced into Lactobacillus casei under the condition of electrotransformation.The currently sold BVDV vaccine is generally expensive and has poor effect.In this study,through gene recombination,the L.casei UPP gene was knocked out to obtain a recombinant strain of L.casei that can express BVDVE2 protein.After late PCR identification and Western Blot identification,we obtained a strain that can expressBVDV E2 protein.Recombinant strain of Lactobacillus casei.Finally,we tested the protective effectiveness of recombinant Lactobacillus casei at the individual level,and challenged the mice of each group on the 23 rd day after the initial immunization.The group had better protection,and to some extent confirmed the possibility of successful recombinant colonies.To make it function as a new type of vaccine,a recombinant strain with effective protection has been screened.In short,this study used genetic recombination to construct a recombinant strain of Lactobacillus casei expressing BVDV E2 protein,and proved its own protective effect,which a brand-new exploration in the field of BVDV vaccine research,Provides a new idea for the development of BVDV live vector vaccine.